Maspin is a unique member of the serpin family with diverse functions. Genetic studies by my laboratory using maspin transgenic and knockout mice demonstrated an important role of maspin in normal mammary and embryonic development. Recently, we have identified several new properties of maspin. As a protein that is present on cell surface, maspin takes part in controlling cell-ECM adhesion. We believe that this function is responsible for maspin-mediated suppression of tumor cell motility and invasion. We have also discovered that maspin is involved in the induction of tumor cell apoptosis through a mitochondrial death pathway. The long-term objectives of our studies are to elucidate the molecular mechanism of maspin action in cell adhesion and apoptosis, and to understand the effect of maspin on normal mammary gland development and breast tumor progression. We have now generated floxed maspin mice for conditional and cell-specific deletion of maspin in order to study the effect of loss of maspin in early mammary development and in myoepithelial cells. We believe that these analyses, especially the study of myoepithelial cells, will offer enormous hope for discovering important information for breast cancer prevention. In this renewal, we propose to combine in vivo mouse genetics with in vitro analyses to study maspin-mediated biological functions, with the following specific aims: Specific Aim 1: To define the role of maspin in mammary ductal development and early breast cancer lesions in vivo and in mammary morphogenesis in vitro. A. To define the role of maspin in mammary ductal development and preneoplastic lesions using maspin conditional knockout mice. B. To study the role of maspin in mammary morphogenesis and epithelial cell polarity, using 3D cell culture models. C. To study the role of myoepithelial maspin in the maintenance of luminal cell polarity in a 3D co- culture model. Specific Aim 2: To dissect the molecular mechanism of maspin-mediated cell adhesion. A. To map the domain(s) and sites in maspin molecule that are responsible for the increased cell adhesion. B. To dissect maspin-mediated signal transduction pathway in cell adhesion. Specific Aim 3: To study the molecular mechanism of maspin action in tumor cell apoptosis. A. To study the role of maspin in mitochondrial-mediated apoptosis. B. To determine whether maspin mediates the cross-talk between ER-dependent and mitochondria-dependent apoptosis. C. To identify other maspin-interacting proteins in cytoplasm, mitochondria, and ER. Cancer Relevance: Maspin is a serpin with tumor suppressing activity. Understanding the molecular mechanism of maspin action not only will help us explain maspin's multiple biological functions in vivo, but also may lead to better means to inhibit breast tumor growth and metastasis.